A gephyrin-related mechanism restraining glycine receptor anchoring at GABAergic synapses

Abstract

Spinal cord neurons release glycine and GABA and accumulate glycine receptors (GlyRs) and GABA(A) receptors in the same postsynaptic densities. In contrast, supramedullar neurons prefer GABA as a neurotransmitter and exclude GlyRs from postsynaptic anchoring. The general aim of the present study was to elucidate the mechanisms underlying transmitter-appropriate receptor accumulation at inhibitory synapses. Specifically, we intended to clarify the molecular basis for the prohibition of GlyR accumulation in the postsynaptic densities of GABAergic synapses. A green fluorescent protein (GFP)-tagged gephyrin-binding loop of the GlyR beta subunit (GFP::betaL) was used as a surrogate for full-length receptors to characterize the GlyR binding capacity of postsynaptic gephyrins in transfected neurons. Both in spinal cord neurons (SCNs) and hippocampal neurons (HNs) GFP::betaL distribution displayed transmitter specificity; i.e., postsynaptic accumulation of GFP::betaL was high opposite terminals able to release glycine and low opposite purely GABAergic terminals. When comparing SCN and HN cultures we found that the level of mRNA coding for gephyrin splice variants containing the cassette C5 (C5-gephyrins) was significantly higher in HNs. In HNs depleted of C5-gephyrins, both GFP::betaL and endogenous GlyRs accumulated at postsynaptic GABAergic sites. Accordingly in SCNs, GFP-tagged C5-gephyrin displayed a preferential postsynaptic accumulation opposite GABAergic synapses. Comparison of glycinergic, mixed, and GABAergic synapses in SCNs showed that the degree of GlyR accumulation was inversely related to the amount of postsynaptic C5-gephyrin. These results identify the C5 splice variant of gephyrin as a factor regulating the transmitter-appropriate degree of GlyR accumulation at inhibitory synapses.

Figure 1.

Gephyrin structure. A, Gephyrin is composed of two domains that are homologous to the bacterial proteins MogA and MoeA and separated by a linker region. The N-terminal domain is involved in trimer formation, whereas the C-terminal domain mediates dimerization and is required for GlyR binding (Rees et al., 2003). Together, both domains are presumably involved in the assembly of an ordered hexagonal lattice. B, The initially identified clone P1 (Prior et al., 1992) bearing cassettes C2 and C6. Insertion of at least six distinct cassettes generates a large number of alternatively spliced gephyrin isoforms. Numbering identifies amino acid positions within the gephyrin open reading frame. N and C termini are indicated.

Figure 5.

RT-PCR analysis of gephyrin expression in neuronal cultures from HNs (DIV8) and SCNs (DIV12) and C5 antisense oligonucleotide design. A, B, Gephyrin isoform expression in HNs (A) and in SCNs (B). C, D, Quantification of the relative amounts of the gephyrin splice variants containing cassettes C5 (C) or C3 (D). Note that a C2 + 5/C2 ratio of 1 is obtained when half of the C2-containing gephyrins contain C5. A C3 to w/o ratio of 1 is obtained when equal amounts of gephyrin contain or lack C3. Compared with SCNs, HNs express significantly more gephyrin with C5 and C3. E, Scheme of the structure of a gephyrin molecule containing cassettes C2, C5, C4, and C6. Increased magnification of the sequence of interest and design of C5-gephyrin sense and antisense phosphorothioate oligonucleotides is shown below. The amino acid sequence is shown in single-letter code. Both oligonucleotides were at the 5′-end TRITC label to allow identification of sense/antisense-transfected neurons. F, RT-PCR analysis of C5 antisense-transfected HNs. Note the dose-dependent depletion of C5-gephyrin transcripts. G, Quantification of the ratio of transcripts with C2 + C5 versus transcripts with C2 only (top panel) and concomitant increase in the amount of C3-containing transcripts (bottom panel).

Figure 2.

Accumulation of transfected GFP::βL opposite inhibitory synaptic terminals in DIV12 SCNs. A, B, COS-7 cells transfected with DsRed2::βL (A) and GeC2,6::GFP (B). DsRed2::βL is trapped by intracellular gephyrin aggregates and therefore can be regarded as capable of binding to gephyrin. C, Image of a neuron containing GFP::βL. D, E, Corresponding endogenous VIAAT (D) and gephyrin (E) immunoreactivity. F, Fraction of VIAAT-immunoreactive terminals that also contained GFP::βL (left y-axis) and fraction of VIAAT- and GFP::βL-immunoreactive synapses that also contained gephyrin (right y-axis). Note the high degree of colocalization between GFP::βL, VIAAT, and gephyrin. Scale bars, 10 μm.

Figure 3.

Transmitter-appropriate postsynaptic accumulation of transfected GFP::βL in DIV12 SCNs. A, B, Same view field. GlyT2 and GAD65 immunoreactivity of terminals (A) making contact with a GFP::βL expressing neuron (B). C, Fraction of glycinergic (GlyT2+ only), mixed (GlyT2+ and GAD65+), or GABAergic (GAD65+ only) terminals facing GFP::βL clusters. D, High-power views of the region delineated in A. Arrowheads show lack of GFP::βL accumulation opposite GAD65-only synapses. E, High-power views of a dendrite taken from another transfected neuron illustrating accumulation of GFP::βL opposite GlyT2-only synapses (crossed arrows). Note that GFP::βL colocalizes with glycinergic or mixed terminals (arrows, crossed arrows) but not with purely GABAergic terminals (arrowheads). Scale bars: A, B, 5 μm; D, E, 2 μm.

Figure 4.

Lack of postsynaptic GFP::βL accumulation in DIV8 HNs. A, Triple immunofluorescence of VIAAT (A1), GAD65 (A2), and GlyT2 (A3). Note lack of GlyT2 immunoreactivity. B, Example of an HN expressing endogenous GlyRs (mAb4a) that were partially colocalized with VIAAT-containing terminals. Inset in B shows the presence of mRNAs coding for GlyR α2 and β subunits. Top bands in each lane represent β-actin amplification products. C, Distribution of transfected GFP::βL. Note the diffuse cytosolic localization of GFP::βL and its absence opposite GAD65-labeled terminals (arrowheads). Scale bars, 10 μm.

Figure 6.

C5-gephyrin depletion in DIV8 HNs allows GFP::βL and endogenous GlyRs to accumulate opposite GABAergic terminals. A, Localization of VIAAT immunoreactivity (red), GFP::βL (green), and gephyrin (blue) in a C5 antisense co-transfected HN. Inset shows the TRITC signal obtained from the co-transfected C5 antisense oligonucleotide. When C5 antisense (50 nm) is present, aggregates of GFP::βL can be found opposite GABAergic terminals (arrows), in addition to some diffuse GFP::βL signal. Note that postsynaptic GFP::βL aggregates are colocalized with endogenous gephyrin. The white box in A delineates the region shown at higher magnification. B, Fraction of VIAAT-immunoreactive terminals that were colocalized with GFP::βL is dependent on the concentration of the C5 antisense oligonucleotide (5, 50, and 500 nm). C, Experiment with 50 nm C5 antisense oligonucleotide. Left ordinate: fraction of VIAAT-immunoreactive spots with colocalized GFP::βL. Right ordinate: fraction of VIAAT and GFP::βL containing synapses that colocalized with endogenous gephyrin. D, Image illustrating the effect of C5 antisense transfection on endogenous GlyR (mAb4a) distribution (inset, TRITC fluorescence). Note the invasion of almost all of the GABAergic synapses with endogenous GlyRs. E, Quantification of the effect of C5 sense and C5 antisense transfection on postsynaptic GlyR accumulation. Scale bars, 10 μm.

Figure 7.

C5-gephyrin predominantly accumulates opposite GABAergic synapses in DIV12 SCNs. A, Immunostaining of GlyT2, GAD65/67, GeC2,5,6::GFP and GlyR α1. B, Quantification of postsynaptic GeC2,5,6::GFP accumulation opposite purely glycinergic (GlyT2), mixed, and GABAergic terminals (GAD65/67). C, Fraction of GeC2,5,6::GFP-containing glycinergic, mixed, or GABAergic synapses that also contained endogenous GlyRs (mAb2b). D, Size of GeC2,5,6::GFP clusters at glycinergic, mixed, and purely GABAergic terminals. E, Quantification of mean fluorescence intensity of GeC2,5,6::GFP at synapses of different type. Scale bar, 2 μm.

Figure 8.

Scheme to illustrate the proposed mechanism for C5-gephyrin-dependent control of GlyR anchoring. A, Postsynaptic lattice containing a variable amount of C5-gephyrin trimers (black). The highest amount is present in purely GABAergic synapses. The suppression of GlyR accumulation may either be limited to the C5-gephyrin sites (B) or additionally involve the area occupied by neighboring C5-lacking gephyrins, provided the latter are also under the influence of C5-gephyrins.